Biomolecular arrays (such as DNA or RNA arrays) are known and are used, for example, as diagnostic or screening tools. Such arrays include regions of usually different sequence biomolecules (such as polynucleotides or polypeptides) arranged in a predetermined configuration on a substrate. These regions (sometimes referenced as “array features”) are positioned at respective locations (“addresses”) on the substrate. Biomolecular arrays typically are fabricated on planar substrates either by depositing previously obtained biomolecules onto the substrate in a site specific fashion or by site specific in situ synthesis of the biomolecules upon the substrate. The arrays, when exposed to a sample, will undergo a binding reaction with the sample and exhibit an observed binding pattern. This binding pattern can be detected upon interrogating the array. For example all biomolecule targets (for example, DNA) in the sample can be labeled with a suitable label (such as a fluorescent compound), and the label then can be accurately observed (such as by observing the fluorescence pattern) on the array after exposure of the array to the sample. Assuming that the different biomolecule targets were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more components of the sample.
In use, the surface of the array is contacted with a solution containing the sample. The speed and specificity of the binding reaction is dependent on several factors, including composition of the solution (ionic strength, pH, polarity, concentration and identity of the sample), temperature, and speed of mixing of the sample. Samples tend to be expensive, precious, or limited to very small quantities. Therefore, current methods seek to reduce the amount of sample required by reducing the amount of sample solution needed to contact the array. One current method accomplishes this by confining the solution under a coverslip placed on top of the array, creating a thin layer of solution between the array surface and the coverslip. While this technique minimizes the quantity of solution required to contact the U.S. Pat. No.array, it eliminates the ability to mix or stir the solution while the array is being exposed to the solution. Mixing is thus limited to diffusion of the sample molecules within the thin layer of solution between the coverslip and the array surface. This results in very long incubation time, typically, over night and up to 24 hours. The coverslip method also does not allow one to seal the system (undesirable, because it allows evaporation at the edges to occur). The coverslip method frequently results in spatially non-uniform binding because of variations in the flatness of the glass, the bending of the glass, and the thickness of the thin layer of solution. The coverslip method is also messy and clumsy to use; during the disassembly process, it is easy to scratch the array since the glass cover is in close contact to the array substrate.
As an alternative, some array manufacturers have created packages for their arrays. In one type of package, the array substrate is glued in place and the package has a sealed inlet and outlet for the liquid sample. These packages usually have a relatively large (compared to the coverslip systems) distance between the array surface and the mating opposite surface used to seal the chamber. This allows the sample solution to flow across the array when injected into the package. The package usually has to be oriented so that the array surface is vertical to allow the leading air bubble to float to the top and out. The sample volumes in these packages are much larger than the coverslip method, typically greater than 100 microliters and up to 500 microliters or more.
Another technique to create an assay chamber for an array is to place a gasket between the array surface and a mating opposite surface and clamp with an external force. The distance between the two surfaces is typically between 0.5 mm and 1.0 mm. This distance is required to allow the sample solution to flow in the chamber without being restricted by capillary forces. An array enclosed in a package having an assay chamber is easier to handle and less likely to be damaged during use because the mating surface is kept at a distance from the array surface. Mixing of the sample solution across the array surface is possible in the assay chamber by either pumping the liquid sample back and forth across the array or rotating the package to move the liquid position within the sealed chamber. The problem with these types of chambers is the large volume of liquid sample required to fill the volume between the two surfaces while covering the array area. Large sample volumes are sometimes not possible or require dilution of the sample to fill the volume. Dilution of the sample reduces sensitivity of the measurement and may extend the incubation time.
Ideally, one would like to approach the small volumes of the coverslip method while allowing for a more protected sealed system. One such system is described in U.S. Pat. No. 6,361,486 to Gordon and U.S. Pat. No. 6,309,875 to Gordon. This technique uses variable orientation centrifugation to move the sample in a thin cross section between the array surface and the back plate. This technique uses centrifugation of the assay chamber to overcome capillary forces that deter mixing of the sample solution. By changing the orientation of the array during the centrifugation, the sample is moved across the array and allowed to mix during incubation. This system requires a reliable seal between the array surface and the back plate that is sufficiently thin to allow small volumes of sample to cover large areas of the array.
To form a good seal, typically a compliant material is compressed between the two surfaces. It is difficult to find compliant material that is sufficiently thin and compatible with the chemistry used for these biochemical experiments. Normal rubber sheet material is much thicker than what is required for this application. To reduce the volume of sample, a gasket thickness of 0.001″ to 0.003″ is required. Sheet materials typically become too flimsy or are relatively difficult to manipulate at such a small scale. One available material is thin sheets (down to 0.002″) of silicone rubber. This material can be cut into the desired shape and placed on the array surface. A back plate is then carefully set in place, and pressure is applied to seal the assay chamber. In practice, this works, but the gasket is delicate, difficult to handle, and hard to keep in place while assembling the apparatus. Adhesives can be applied to one side of the silicone sheet. This allows the thin sheet of silicone rubber to be applied to the back plate and cut to the desired shape. Unwanted areas of the sheet are then peeled away. The sheet of silicone rubber can also be die cut to form the gasket before it is applied to the back plate. This is a difficult process. The adhesive adds to the thickness of the gasket and has to be compatible with all the chemicals that might be used in the biochemical assay. The silicone sheet material that forms the gasket must be wide enough (on the order of 1+millimeters) to provide strength and structural integrity to survive the process of applying the material to the plate. Therefore, while creating a chamber on the order of about 0.002″ thick is possible using thin silicone sheet material, it is inconvenient and results in relatively wide strips of sheet material on the surface of the back plate (or, alternatively, the surface of the substrate).
There is thus a need for an array system allowing the use of relatively small amounts of sample solution while allowing the sample solution to be mixed or moved across the surface of the array to speed the binding reaction. Such an array system needs to have an assay chamber that is fluid tight to allow the sample solution to flow across the surface of the array and to be mixed without leaking.